Drug seeking and reward can be characterized by the burst firing of dopamine (DA) neurons in the ventral tegmental area (VTA) and increased DA release within the mesocorticolimbic pathway. Other neurotransmitters, such as acetylcholine (ACh), act in the VTA to increase DA release in an area of the pathway called the nucleus accumbens (NAc). ACh also mediates the behaviorally reinforcing characteristics of drugs of abuse. Our lab recently found that methamphetamine (MA) increases ACh and DA in VTA. The VTA primarily receives ACh connections from a mesopontine region known as the laterodorsal tegmentum (LDT) that when active can increase DA activity and induce release in other regions of the pathway. Thus, LDT cholinergic neurons may be involved in MA-induced increases in DA and ACh. Further, the intra-VTA ACh tone may mediate MA-seeking behaviors. The goals of this research involve isolating the ACh projection from the LDT to the VTA in order to determine its importance in the neurochemical (Specific Aim 1) and behavioral (Specific Aim 2) effects of MA in mice. The first aim will determine the involvement of the LDT ACh connection on MA-induced increases in ACh and DA. In this experiment I will reversibly inactivate the LDT ACh connection to the VTA and use microdialysis to measure DA and ACh in the VTA of mice. I anticipate that the inactivation of the LDT ACh will block MA-induced increases in ACh and DA in the VTA. The second aim will determine the involvement of the LDT ACh input to the VTA on reinstatement to MA-seeking. In this experiment I will train mice to self-administer MA and then extinguish this behavior. Typically after extinction a small priming dose of MA will reinstate MA- seeking behavior. Before this MA priming dose is administered I will reversibly inactivate the LDT ACh connection. I anticipate this inhibition of ACh will block MA-primed reinstatement to drug seeking. Persistent drug-seeking characteristic of MA addiction is maintained by the interactions of a wide neurochemical milieu within the mesocorticolimbic pathway. Although ACh has been found to be critically involved in reward and MA-related behaviors, the role of the LDT ACh projections to the VTA in MA-seeking behavior has yet to be characterized. This proposal combines neurochemical and behavioral approaches to define how this cholinergic projection affects the neurochemical environment within the VTA and ultimately affect MA-seeking. PUBLIC HEALTH RELEVANCE: A particularly vulnerable phase of methamphetamine addiction is following a period of abstinence when a priming dose of the drug or exposure to previously drug-paired stimuli (cues) can induce relapse. In order to develop more effective treatment strategies, it is important to understand the neurochemical underpinnings of reinstatement to methamphetamine-seeking behaviors. This proposal will use a combination of behavioral and neurochemical techniques to determine the neurochemical substrates that drive methamphetamine seeking.